A monoclonal antibody as active pharmaceutical ingredients of an antibody drug containing an antibody as a main component is mainly expressed in a culture fluid as a recombinant protein using a mammalian cultured cell or the like, and purified to a high purity by several steps of chromatography and filtration process before formulation.
An antibody drug includes not only a molecule generally called an antibody such as immunoglobulin G and an analog thereof, but also an Fc fusion protein (Fc-containing molecule) in which an Fc region of a constant region of an immunoglobulin molecule is fused to another functional protein or peptide. Further, antibody drugs include low-molecular antibodies such as Fab, scFv, and diabody. Antibody drugs are also prepared by purifying and formulating from recombinant microorganisms, secreted substances in the culture supernatant, or expressed substances in bacterial cell or periplasmic space.
Impurities such as aggregates of antibodies (a dimeric and multiple form of a monomer) which are formed or remains in the steps of culture, purification and formulation is a major cause of side effects, and it is an important issue to reduce the impurities on production of an antibody preparation. Here, a monomer is defined as a unit of a molecule of an antibody having a tetramer structure composed of two molecules of heavy chains (H chains) consisting of an Fc region of a constant region, and a variable region, and two molecules of light chains (L chains) consisting of a variable region. A multimer of the unit molecules is regarded as an aggregate, and thought to be a major cause of side effects of an antibody preparation.
Attempts to control suppression of production of the aggregate and remove the aggregate have been made by a complicated management technique and use of an additive in the steps of culture, purification and formulation. Especially, not only suppression of production of the aggregate, but also removal of the aggregate is important in the purification step. Thus, development of a simple and efficient technique for removing the aggregate has been required in the purification step.
Patterning of purification techniques by combining particular unit operations (making of a platform) is developed in the purification step of the antibody preparation. In the early purification step (recovery step), an antibody affinity separation matrix in which protein A is immobilized as a ligand on a water-insoluble carrier (protein A carrier) is widely utilized. A technique of adsorbing an antibody to the protein A carrier under neutral conditions, and eluting the antibody under acidic conditions is generally used. In general, the antibody is purified at high purity with three chromatography steps, and impurities such as an aggregate are removed by a combination of ion exchange chromatography, hydrophobic interaction chromatography and the like, in the subsequent step of protein A chromatography step (Non-patent Document 1, Non-patent Document 2, Non-patent Document 3, Patent Document 5).
An affinity ligand has a function of specifically binding to a particular molecule, and an affinity separation matrix (hereinafter referred to as affinity chromatography carrier, or affinity carrier) prepared by immobilizing the ligand to a water-insoluble carrier is utilized for efficient separation and purification of a useful substance from biological components or recombinant cell culture including microorganisms and mammalians. An industrially utilized antibody affinity ligand includes, for example, a peptide ligand or a protein ligand derived from a microorganism such as protein A, protein G and protein L or consisted of a functional variant (analog substance) obtained by recombinant technology thereof; a recombinant protein ligand such as a camel single strand antibody and an Fc receptor of an antibody; and a chemosynthetic ligand such as a thiazole derivative. The antibody affinity ligand is used in purification of an antibody drug and the like. Since the antibody preparation has lower toxicity and higher specificity than chemicals, there is much demand for an antibody drug as an ideal pharmaceutical.
In the separation and purification of antibodies from the affinity carrier, there is a problem to remove the aggregate of antibodies, impurities from host, and degradation products of the antibody (hereinafter referred to as aggregates and the like).
For example, the protein A chromatography step is generally carried out with acidic elution in one example of an affinity carrier. However, since the process design thereof takes much time due to the requirement of different elution pH every antibody and the lower the elution pH is, the more the risk of formation of an aggregate is, the protein A ligand is modified by means of protein engineering, so that the antibody which requires pH elution as low as about pH 3 can also be eluted near pH 3.5 to 4 (Patent Document 1).
Moreover, after protein A chromatography step, high content of the aggregate is resulted in lowering of yield of the objective monomeric substance (monomer) in the subsequent step of removing impurities. Thus, not only suppression of the formation of an aggregate, but also removal of an aggregate is studied in the protein A chromatography step.
In addition, a method for decreasing impurities such as aggregates is examined in the protein A chromatography step. That is to say, optimization of pH and ionic strength at the time of elution, as well as fractionation of the first half of the elution peak and the second half of the elution peak, and the like are proposed.
Concretely, there are methods utilizing slight decrease of dissociation constant by contacting an antibody molecule polymerized with a protein A ligand with higher frequency than that of an antibody molecule which is not polymerized as a characteristic of the protein A carrier, and utilizing separation mechanisms based on delicate adjustment of hydrophobicity (Patent Document 2, Patent Document 3, Patent Document 4). However, since these methods are difficult to be strictly controlled and have low resolution, these methods are not used as a general separation technique, and the removal of impurities is required in the subsequent steps.
The affinity chromatography represented by protein A chromatography uses an acidic pH for elution, and the subsequent ionic exchange chromatography and hydrophobic interaction chromatography and the like generally use pH of 5.0 or more, so that the adjustments of pH and ionic strength are required.
On the other hand, the cation exchange carrier is generally used at higher pH than pKa of a ligand thereof. The adsorption and desorption to the cation exchange carrier for a target protein is performed at lower pH than isoelectric point (pI) of the protein. For example, in the purification of antibodies having pI of 8, the cation exchange carrier having a sulfone group having pKa of about 2 or a carboxyl group having pKa of about 3 to 5 as a ligand is used both with the buffer having pH of 5 to 6 to adsorb and desorb for the purification of target proteins. The pH of the buffer is set between pKa of the ligand and pI of the target proteins. In the case where the used pH is far lower than pI, the positive charge of the proteins is increased, and high ionic strength is necessarily set for the elution, so that it is likely to decrease the recovery. In the case where the ionic strength of the eluate is high, the ionic strength sometime has to be decreased in the subsequent process constructions. In addition, when the used pH is near or lower than pKa of the cation exchange ligand, the negative charge of the ligand is protonated and the binding capacity of the ligand is decreased, so that such low pH range generally has not been selected. Therefore, the buffer having pH of 5 to 6 has been used for the cation exchange carrier having pKa of 2 to 5 in the purification of antibodies using the cation exchange carrier.
Not only when the anion exchange chromatography step or the hydrophobic interaction chromatography step is used after the affinity chromatography step (Patent Document 8), but also when the anion exchange chromatography step is performed after the cation exchange chromatography (Patent Document 7) or the multiple target substances are collected at the cation exchange chromatography step (Patent Document 6), the adjustments of pH and ionic strength have been required.
In the case where the antibodies and the like are purified with the affinity chromatography and are further purified at high degree in the subsequent processes, the adjustments of pH and ionic strength of acidic eluate are required and there are limitations for the efficiency of continuous chromatographies initialized. In addition, even when the ionic exchange chromatography step and the hydrophobic charge induction chromatography step are performed without using the affinity chromatography purification, there were limitations for the efficiency that each step must be performed independently and the antibodies cannot be purified continuously (Patent Document 9).
In addition, although the antibody affinity separation matrix exhibits high specificity to an antibody and can collect the antibody at high purity, the ability of separating a monomeric substance (monomer) and an aggregate and the like is low even if the usage is strictly set. Thus, the antibody affinity separation matrix had limitation for removing an aggregate.